DESCRIPTION: (Verbatim from applicant's abstract) The long term goal of this project is to understand how cells regulate the transport and assembly of gap junctions. Newly synthesized connexins are assembled into hexamers in an intracellular compartment prior to the formation of gap junction channels. We found that ROS cells differentially assemble and sort two endogenously expressed gap junction proteins, CX43 and CX46. ROS cells assemble CX43 into hexamers which are transported to the plasma membrane. In contrast, CX46 remains a monomer and is restricted to the trans Golgi network (TGN). The goal of this proposal is to understand how CX43 and CX46 are differentially regulated by these cells. To identify domains that control connexin targeting, we will make epitope tagged connexin chimeras containing domains from CX43 and CX46. These will be transfected into cell lines and then analyzed by immunofluorescence microscopy for transport to the plasma membrane and/or TGN. These constructs will then be analyzed by sucrose gradient fractionation of Triton X-100 solubilized cell extracts to determine whether all TGN localized connexins are retained as monomers. We will then determine whether Cx43 and Cx46 can form hybrid gap junction channels. This is a key point-the compatibility of these connexins to form hybrid channels would imply that there is an active mechanism to keep them from co-assembling in ROS cells. Hybrid CX43+CX46 channels formed during in vitro translation or by Hela cells where both proteins are transported to the plasma membrane will be solubilized as intact hexamers and identified by co-immunoisolation. Finally, we will determine whether Cx46 monomers co-exist with Cx43 hexamers in the same intracellular compartments. Cells will be treated to arrest connexin transport at different steps, homogenized and intact vesicles containing CX46 will be immunoisolated and then analyzed for the presence of CX43 hexamers. Understanding the fundamentals of differential CX43 and CX46 sorting and assembly will provide insights into the regulation of gap junctional communication and is likely to reflect general principles of protein assembly in post-Golgi compartments.